This invention relates generally to the field of body fluid analysis and more particularly to an improved method and apparatus for analyzing multiple samples, in-vitro, utilizing radioactive tracers to determine relative resin binding of the constituent to be determined.
An application of the invention relates to an improved method and apparatus for determining the thyroid hormone content of blood. Thyroid hormones, such as triiodothyronine, or T.sub.3 as it is commonly known, are produced in the thyroid gland and are carried in the blood to the body cells where the hormones ultimately produce their well-known metabolic effects. The thyroid hormones regulate the activity of human cells in carrying out the particular functions of these cells. Consequently, when the thyroid gland puts out an excess of these hormones, these body cells are overactive, the body burns up excess food and the patient has the condition known as hyperthyroidism. In hyperthyroidism, the reverse situation caused by a deficiency of thyroid hormones takes place.
It is well known that the thyroid hormones do not exist freely in large amounts in the plasma but are bound to specific protein fractions of that tissue. In this bound form, the hormones are transported throughout the body. The thyroid hormones are specifically bound to two distinct plasma proteins, thyroxine binding globulin (TBG) and prealbumen, which are normally present in such low concentrations as to defy analysis by conventional methods. Nevertheless, the binding strength or capability of the protein fractions to bind the thyroid hormones and the quantity of binding proteins available has been shown to be generally constant within narrow limits in various blood samples from most humans.
It is also well known that certain resins also have the ability to bind the thyroid hormones. In other words, these resins have an affinity for the thyroid hormones so that in the presence of these well known resins, a fraction of the thyroid hormones becomes bound to the resins even in the presence of serum proteins. The magnitude of this fraction is influenced primarily by the quantity of thyroid hormone in the blood when the binding capacity of thyroxine binding proteins is within normal limits. Consequently, it has been the practice to test indirectly for the thyroid hormone content of blood by contacting the blood with a radioactive thyroid hormone, and a quantity of a resin having an affinity for the thyroid hormone. In the case of a hyperthyroid, in which the thyroid gland of the patient was overactive, the protein fraction in the serum approaches saturation with thyroid hormones. Consequently, the resin attracts and binds a large proportion of the radioactive thyroid hormone. The extent to which the resin takes up radioactive hormone from the blood sample is an indirect measure of the thyroid hormone content of the blood sample. This measurement is sometimes simply stated at "T.sub.3 resin uptake," so in hyperthyroidism there is a high T.sub.3 resin uptake. In a hyperthyroid situation, the amount of thyroid hormone present in the patient's serum is reduced so that the serum proteins are "starved" for hormones. As a result, more of the added radioactive hormone will be bound to plasma proteins and less to the resin having an affinity for the hormone. Consequently, the test, in such a situation, will show a lesser take-up of hormone by the resin, i.e., a low T.sub.3 resin uptake.
In carrying out the above test, the usual procedure is to obtain serum from the patient, place this serum in a test tube or glass vial, add a predetermined amount of resin to the vial, and then add radioactive T.sub.3 to the serum-resin mixture. In one method the T.sub.3 is added directly to the resin. The radioactivity of this mixture is recorded and then the mixture is incubated for a period of time between thirty minutes and two hours. In many methods agitation or mixing is required during this incubation period. After the incubation period, the resin must be separated from the serum, following which the amount of radioactive material bound to either the serum alone or the resin alone is determined by making another radioactivity recording. This separation is generally accomplished by allowing the resin to settle (by centrifugation or by gravity) and either (a) withdrawing a known fraction of the overlying serum, or (b) washing the settled resin by adding several volumes of water decanting the water-serum mixture after each addition of water. The long incubation period with agitation was thought to be necessary for accurate analysis since that represented the approximate time for the system to reach equilibrium and the resin to absorb the maximum possible amount of hormone.
Similarly, Murphy and Pettee described a procedure for the determination of T.sub.4 as thyroxine by competitive protein-binding analysis utilizing radioactive thyroxine which is allowed to interact with the patient's serum in the presence of a stabilizing agent and then separating the unbound thyroxine from the bound thyroxine by means of an ion exchange resin which competes for the thyroxine with the thyroxine binding globulin (TBG) of the blood. Still another method is described for T.sub.3 analysis in U.S. Pat. No. 3,451,777, wherein an excess of resin is used in a fixed, packed column, which is washed by forcing a wash fluid through the column which has been contacted with a sample-radioactive T.sub.3 mixture.
The purpose of this invention is to provide a method which enables good precision, sensitivity and range of reportable value, while, in addition, providing a method and device which will markedly aid patient testing by enabling multiple samples and controls to be processed for analysis at essentially the same time, for shorter periods of time, and in a time controlled manner.